The present invention relates to a process of coding and identifying individual members of a chemical combinatorial library synthesized on a plurality of solid supports. The process provides for tagging the solid supports with a coding identifier that is decoded while attached to the solid support by infrared or Raman spectroscopy.
Typically, methods for the synthesis of large numbers of diverse compounds involve successive chemical modifications of existing molecules. Modifications can include the addition of a chemical unit to a growing sequence or modification of a functional group. Chemical units can take many forms, both naturally-occurring and synthetic, including compounds containing reactive functional groups such as neculeophiles, electrophiles, dienes, alkylating agents, acylating agents, diamines, nucleotides, amino acids, sugars, lipids or derivatives thereof, organic monomers, synthons, and combinations thereof. Alternatively, reactions can be involved which result in alkylation, acylation, nitration, halogenation, oxidation, reduction, hydrolysis, substitution, elimination, addition, and the like. This process can produce non-oligomers, oligomers, or combinations thereof in extremely small amounts, where the reaction history, and composition in appropriate cases, can be defined by the present tags. Non-oligomers include a wide variety of organic molecules, e.g., heterocyclics, aromatics, alicyclics, aliphatics and combinations thereof, such as steroids, antibiotics, enzyme inhibitors, ligands, hormones, drugs, alkaloids, opioids, terpenes, porphyrins, toxins, catalysts, as well as combinations thereof. Oligomers include oligopeptides, oligonucleotides, oligosaccharides, polylipids, polyesters, polyamides, polyurethanes, polyethers, poly (phosphorus derivatives) e.g., phosphates, phosphonates, phosphoramides, phosphonamides, phosphites, phosphinamides, etc., poly (sulfur derivatives) e.g., sulfones, sulfonates, sulfites, sulfonamides, sulfenamides, etc., where for the phosphorous and sulfur derivatives the indicated heteroatom for the most part will be bonded to C, H, N, O and combinations thereof.
Reactions can involve modifications at a variety of random sites of a central core molecular structure or modifications at specific sites. For example, one can brominate a polycyclic compound, where bromination can occur at a plurality of sites or use a brominating agent which will be specific for a particular site, e.g., N-bromosuccinimide. Typically, reactions will involve single sites or equivalent sites, for example, one of two hydroxyl groups of a glycol. For the most part, the subject synthesis will have at least two stages where other than bifunctional compounds are attached using the same linking functionality, e.g., amino acids and amide bonds, nucleotides and phosphate ester bonds, or mimetic compounds thereof, e.g., aminoisocyanates and urea bonds.
The synthetic strategies will vary with the nature of the group of products one wishes to produce. Thus, the strategy must take into consideration the ability to change the nature of the product, while allowing for retention of the results of the previous stages and anticipating needs for the future stages. Where the various units are of the same family, such as nucleotides, amino acids and sugars, the synthetic strategies are relatively well-established and frequently conventional chemistry will be available. Thus, for nucleotides, phosphoramidite or phosphite chemistries can be employed; for oligopeptides, fluorenylmethyl (Fmoc), t-butyoxycarbonyl (Boc), etc. protection chemistries can be employed; for sugars, the strategies can be less conventional but a large number of protective groups, reactive functionalities, and conditions have been established for the synthesis of polysaccharides. For other types of chemistries, one will look to the nature of the individual unit and either synthetic opportunities will be known or will be devised, as appropriate.
Techniques have recently been developed wherein individual units are added sequentially in a controlled or random manner to produce all or a substantial proportion of possible compounds resulting from the different choices possible at each sequential stage in the synthesis. One disadvantage is that individual compounds are present only in minute amounts. While the biological activity of a given compound can be determined, the chemical structure of that particular compound cannot necessairly be determined. It is necessary for compounds made by such techniques to be amenable to methods for determining their composition.
There is a substantial interest in discovering methods for producing compounds which are not limited to sequential addition of like moieties, but frequently involve a multi-stage synthesis in which the reagents and/or conditions are varied to provide a variety of compounds. There needs to be, however, convenient ways to identify the structures of the large number of compounds which result from a wide variety of different modifications. Thus, there is a need to record the reaction history or the structures of the compound identified.
As the size of compound libraries increases, existing means for elucidating structure and segregating products introduce substantial inefficiencies and uncertainties that hinder accurate structure determination. Thus, there is a substantial need for new methods which will permit the synthesis of complex combinatorial chemical libraries, which readily permit accurate structural determination of individual compounds within the library which are identified as being of interest.
The present invention provides a process of coding individual members of a combinatorial chemistry library using attached Infrared or Raman chromophores. The process provides for the partial or complete identification of a library member compound or the synthetic pathway generating that compound on any solid support.
The solid support is uniquely tagged to define a particular event, usually chemical, associated with the synthesis of the compound on the support using coding identifier molecules that record the sequential events to which the supporting particle is exposed during synthesis, thus providing a reaction history for the compound produced on the support.
Each coding identifier is stable under the synthetic conditions employed, remains associated with the support during the stage of the synthesis, assay, and library cleavage, uniquely defines a particular event during the synthesis that reflects a particular reaction choice at a given stage of the synthesis, and is distinguishable from other components that can be present during assaying. The coding identifier is covalently attached to the solid support.
By associating each stage or combination of stages (e.g., xe2x80x9cadd reagent Axe2x80x9d or xe2x80x9cadd reagent A, then reagent B, and heat to 100xc2x0 C. for 2 hoursxe2x80x9d) of the serial synthesis with an identifier which defines the choice of variables such as reactant, reagent, reaction conditions, or a combination of these, one can use the identifiers to define the reaction history of each definable and separable substrate. The spectrophotometric analysis of identifiers allows for ready identification of the reaction history, at picomolar or lower concentrations. One can determine a characteristic of a product of a synthesis, usually a chemical or biological characteristic by various screening techniques, and then identify the reaction history and thereby the structure of that product, which has the desired characteristic, by virtue of the tag(s) associated with the product.
An advantage of the present invention lies in the fact that the code can be read directly on the bead, thus expanding the scope of chemistry compatible with the tag as well as the scope of assay capabilities: the tag(s) remains with the bead during partial or complete release of the library entity.
The use of the instant multiple tag system avoids the necessity of carrying out a complicated cosynthesis which reduces yields and requires multiple protecting groups and avoids the necessity of using sequential (e.g., nucleic acid or peptide oligomers) tags which are necessarily chemically labile. Both the necessity of multiple protecting groups and the intrinsic instability of all known sequential tagging molecules severely limit the chemistry which can be used in the synthesis of the library element or ligand.
The coding identifiers of this invention are used in combination with one another to form a binary or higher order encoding system permitting a relatively small number of identifiers to be used to encode a relatively large number of reaction products. For example, N identifiers can uniquely encode up to 2N different compounds in a binary code. Thus, 30 distinguishable tags are available and are sufficient to encode  greater than 109 different syntheses. A ternary coding system could encode for this same number with significantly less than 30 distinguishable tags.
Moreover, the use of a binary, or higher, multiple tag system reduces enormously the number of tags necessary to encode the reagent/reactant choice in any stage in a synthesis. For example, if a particular synthetic stage could be carried with 125 different choices for reagent, the binary system would require only 7 tags. Further, ternary coding would accomplish this with substantially less tags. This can make the difference between a practical encoding system and an impractical one, because it may not be feasible to obtain and use the large number of distinguishable tags required by other systems.